Knockdown Of Fyn Research Articles

Fyn Kinases Play a Critical Role in Neuronal Apoptosis Induced by Oxygen and Glucose Deprivation or Amyloid‐β Peptide Treatment

Src family protein tyrosine kinases (SrcPTKs) have been implicated in the pathogenesis of brain ischemia and Alzheimer's disease (AD). In this study, we investigated whether Src and Fyn kinases, two major members of SrcPTKs in the brain, have distinct roles in the oxygen and glucose deprivation (OGD) and amyloid-β peptide (Aβ)-induced neuronal apoptosis. The DAPI staining and caspase-3 activation analysis showed that small interfering RNAs (siRNAs) knockdown of Src or Fyn attenuated SH-SY5Y cells apoptosis after OGD and Aβ treatment. Fyn knockdown had a more potent neuroprotective effect than Src knockdown, suggesting a principal pathological significance of Fyn in brain ischemia and AD. Previously, we reported that brain ischemia promotes the phosphorylation of postsynaptic density protein 95 (PSD-95) at tyrosine 523 (Y523), which is associated with postsynaptic mechanisms of excitotoxicity. Here, immunoblot analysis indicated that not only OGD but also Aβ incubation increased the PSD-95Y523 phosphorylation. Src knockdown, especially Fyn knockdown, significantly inhibited such phosphorylation. Fyn mediates PSD-95Y523 phosphorylation, which may be responsible for the excitotoxic signal cascades and neuronal apoptosis in brain ischemia and Aβ neurotoxicity. Fyn is a potential therapeutic target for the treatment of ischemic stroke and AD.

Myosin II is a negative regulator of oligodendrocyte morphological differentiation

During their development as myelinating cells, oligodendrocyte progenitors (OPC) undergo dramatic changes in the organization of their cytoskeleton. These changes involve an increase in cell branching and in lamella extension, which is important for the ability of oligodendrocytes to myelinate multiple axons in the CNS. We have previously shown that the levels of the actin-associated motor protein nonmuscle myosin II (NMII) decrease as oligodendrocyte differentiate and that inhibition of NMII activity increases branching and myelination, suggesting that NMII is a negative regulator of oligodendrocyte differentiation. In agreement with this interpretation, we have found that overexpression of NMII prevents oligodendrocyte branching and differentiation and that OPC maturation is accelerated in NMII knockout mice as shown by a significant increase in the percentage of mature MBP(+) cells. Although several pathways have been implicated in oligodendrocyte morphogenesis, their specific contribution to the regulation of NMII activity has not been directly examined. We tested the hypothesis that the activity of NMII in OPC is controlled by Fyn kinase via downregulation of RhoA-ROCK-NMII phosphorylation. We found that treatment with PP2 or knockdown of Fyn using siRNA prevents the decrease in myosin phosphorylation normally observed during OPC differentiation and that the inhibition of branching induced by overexpression of constitutively active RhoA can be reversed by treatment with Y27632 or blebbistatin. Taken together, our results demonstrate that Fyn kinase downregulates NMII activity, thus promoting oligodendrocyte morphological differentiation.

Deficiency of Fyn protein is prerequisite for apoptosis induced by Src family kinase inhibitors in human mesothelioma cells

Malignant mesothelioma is an aggressive tumor arising from mesothelial cells of serous membranes. Src family kinases (SFKs) have a pivotal role in cell adhesion, proliferation, survival and apoptosis. Here, we examined the effect of SFK inhibitors in NCI-H2052, ACC-MESO-4 and NCI-H28 cells, mesothelioma cell lines and Met5A, a human non-malignant mesothelial cell line. We found that PP2, a selective SFK inhibitor, inhibited SFK activity and induced apoptosis mediated by caspase-8 in NCI-H28 but not Met5A, NCI-H2052 and ACC-MESO-4 cells. Src, Yes, Fyn and Lyn protein, which are members of the SFK, were expressed in these cell lines, whereas NCI-H28 cells were deficient in Fyn protein. Small interfering RNA (siRNA) targeting Fyn facilitated PP2-induced apoptosis mediated by caspase-8 in NCI-H2052 and ACC-MESO-4 cells. PP2 reduced Lyn protein levels and suppressed SFK activity in all mesothelioma cell lines. Lyn siRNA induced caspase-8 activation and apoptosis in NCI-H28 cells but not in NCI-H2052 and ACC-MESO-4 cells. However, double RNA interference knockdown of Fyn and Lyn induced apoptosis accompanied by caspase-8 activation in NCI-H2052 and ACC-MESO-4 cells. Dasatinib, an inhibitor of multi-tyrosine kinases including SFK, also inhibited SFK activity and induced reduction of Lyn protein levels, caspase-8 activation and apoptosis in NCI-H28 cells but not in other cell lines. Present study suggests that SFK inhibitors induce caspase-8-dependent apoptosis caused by reduction of Lyn protein in Fyn-deficient mesothelioma cells.

The Role of p27 Kip1 in Dasatinib-Enhanced Paclitaxel Cytotoxicity in Human Ovarian Cancer Cells

Less than 50% of ovarian cancers respond to paclitaxel. Effective strategies are needed to enhance paclitaxel sensitivity. A library of silencing RNAs (siRNAs) was used to identify kinases that regulate paclitaxel sensitivity in human ovarian cancer SKOv3 cells. The effect of dasatinib, an inhibitor of Src and Abl kinases, on paclitaxel sensitivity was measured in ovarian cancer cells and HEY xenografts. The roles of p27(Kip1), Bcl-2, and Cdk1 in apoptosis induced by dasatinib and paclitaxel were assessed using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, siRNA knockdown of gene expression, transfection with Bcl-2 and Cdk1 expression vectors, and flow cytometry. All statistical tests were two-sided. Src family and Abl kinases were identified as modulators of paclitaxel sensitivity in SKOv3 cells. The siRNA knockdown of Src, Fyn, or Abl1 enhanced paclitaxel-mediated growth inhibition in ovarian cancer cells compared with a control siRNA. HEY cells treated with dasatinib plus paclitaxel formed fewer colonies than did cells treated with either agent alone. Treatment of HEY xenograft-bearing mice with dasatinib plus paclitaxel inhibited tumor growth more than treatment with either agent alone (average tumor volume per mouse, dasatinib + paclitaxel vs paclitaxel: 0.28 vs. 0.81 cm3, difference = 0.53 cm3, 95% confidence interval [CI] = 0.44 to 0.62 cm3, P = .014); dasatinib + paclitaxel vs. dasatinib: 0.28 vs. 0.55 cm3, difference = 0.27 cm3, 95% CI = 0.21 to 0.33 cm3, P = .035). Combined treatment induced more TUNEL-positive apoptotic cells than did either agent alone. The siRNA knockdown of p27(Kip1) decreased dasatinib- and paclitaxel-induced apoptosis compared with a negative control siRNA (sub-G1 fraction, control siRNA vs. p27(Kip1) siRNA: 42.5% vs. 20.1%, difference = 22.4%, 95% CI = 20.1% to 24.7%, P = .017). Studies with forced expression and siRNA knockdown of Bcl-2 and Cdk1 suggest that dasatinib-mediated induction of p27(Kip1) enhanced paclitaxel-induced apoptosis by negatively regulating Bcl-2 and Cdk1 expression. Inhibition of Src family and Abl kinases with either siRNAs or dasatinib enhances paclitaxel sensitivity of ovarian cancer cells through p27(Kip1)-mediated suppression of Bcl-2 and Cdk1 expression.

Fyn is induced by Ras/PI3K/Akt signaling and is required for enhanced invasion/migration

Src family kinases (SFKs) are frequently over-expressed and/or activated in human cancers, and play key roles in cancer cell invasion, metastasis, proliferation, survival, and angiogenesis. Allosteric activation of SFKs occurs through well-defined post-translational mechanisms, however the SFK member Fyn is over-expressed in multiple human cancers (prostate, melanoma, pancreatic, glioma, chronic myelogenous leukemia) and the mechanism of increased Fyn expression is unclear. Since activation of Ras oncogenes is a common oncogenic event leading to the activation of multiple effector pathways, we explored if Ras could induce Fyn expression. Retroviral transduction of the human keratinocyte cell line HaCaT with oncogenic H-Ras dramatically up-regulated Fyn mRNA (>100-fold, P < 0.001), protein, and kinase activity without affecting Src levels or activity. Activation of Akt, but not MAPK or EGFR, was necessary and sufficient for induction of Fyn by H-Ras. Expression of active Fyn was sufficient to increase HaCaT cell migration and invasion, and the enhanced migration and invasion induced by H-Ras could be significantly blocked (70% reduction, P < 0.01) by knockdown of Fyn with a specific siRNA or inhibition of SFKs with PP2. In addition, expression of Fyn in MDA-MB-231 breast cancer cells was dependent on PI3K activity and was involved in their invasive phenotype. Thus, the Ras/PI3K/Akt pathway can account for Fyn over-expression in cancers, and Fyn is a critical mediator of the Ras-stimulated invasive cell phenotype. These results support the development of therapeutic strategies targeting Akt/Fyn pathway to block migration and invasion of tumor cells.

Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP3)-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP3 or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP3-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP3-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP3-mediated calcium release by phosphorylating Type I IP3 receptors (IP3R1), we investigated the effect of glucocorticoids on IP3R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP3R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP3-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP3-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.

Regulation of peripheral myelination by Src-like kinases

Fyn, a nonreceptor Src-like tyrosine kinase (SLK), plays an important role in oligodendrocyte differentiation and myelination in the brain. However, its role in myelination of peripheral nerves remains undefined. Here we report that selective inhibitors of SLKs (PP2 and SU6656) caused a dose-dependent decrease in the accumulation of several myelin proteins, including myelin basic protein (MBP), protein zero (P0) and myelin-associated glycoprotein (MAG) in rat Schwann cell-dorsal root ganglion neuron (SC-DRGN) co-cultures. Interestingly, SLK inhibition was insufficient to completely abrogate myelin synthesis, as removal of PP2 after several days of treatment permitted a partial recovery of myelin proteins expression. Furthermore, fewer and shorter myelinated segments formed in the continuous presence of PP2, although the myelin formed was normally compacted. PP2 also decreased the number of SCs expressing Krox-20, a master-regulatory transcription factor expressed by myelinating SCs, by 50%. These results were corroborated by selective knockdown of Fyn and Lyn kinases using siRNA. Extracellular matrix is important to SC differentiation and peripheral myelination. Using phospho-specific antibodies, we showed that addition of extracellular matrix extracts to SC-DRGN co-cultures resulted in the activation of ERK, Akt and p38 MAPK, three protein kinases involved in SC proliferation, differentiation and peripheral myelination. PP2 blocked the phosphorylation of all three kinases. Our results support a role for SLKs in the initiation of peripheral myelination via the activation of p38, Akt and ERK, which regulate Krox-20 expression and peripheral myelination.

Dopamine D1 receptor-mediated NMDA receptor insertion depends on Fyn but not Src kinase pathway in prefrontal cortical neurons

BackgroundInteractions between dopamine and glutamate in the prefrontal cortex are essential for cognitive functions such as working memory. Modulation of N-methyl-D-aspartic acid (NMDA) receptor functions by dopamine D1 receptor is believed to play a critical role in these functions. The aim of the work reported here is to explore the signaling pathway underlying D1 receptor-mediated trafficking of NMDA receptors in cultured rat prefrontal cortical neurons.ResultsActivation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits. This effect was completely blocked by small interfering RNA knockdown of Fyn, but not Src. Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression. D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.ConclusionsDopamine D1 receptor-mediated increase of NMDA receptors is thus Fyn kinase dependent. Targeting this signaling pathway may be useful in treating drug addiction and schizophrenia.

Chromium(VI) Stimulates Fyn to Initiate Innate Immune Gene Induction in Human Airway Epithelial Cells

Mechanisms for pathogenic metal signaling in airway injury or disease promotion are poorly understood. It is widely believed that one mechanism for pathogenic and possible carcinogenic effects of inhaled chromium (Cr(VI)) is inhibition of inducible gene transactivation. However, we recently reported that Cr(VI) inhibition of Sp1-dependent transactivation required signal transducer and activator of transcription 1 (STAT1)-dependent expression of an inhibitory protein in airway epithelium. Thus, Cr(VI) exposures can induce genes, and we hypothesized that this induction resulted from Cr(VI) signaling through an innate immune-like STAT1-dependent pathway initiated by Fyn. Exposure of human airway epithelial (BEAS-2B) cells to Cr(VI) selectively transactivated the STAT-responsive interferon-stimulated response element (ISRE) and induced ISRE-driven transactivation of interferon regulatory factor 7 (IRF7), without affecting the gamma interferon-activated site (GAS)-driven IRF1 expression. Cr(VI)-induced IRF7 was absent or greatly reduced in cells that lacked STAT1, were treated with the Src family kinase inhibitor, PP2, or lacked Fyn. Expressing Fyn, but not Src, in mouse embryonic fibroblasts cells null for Src, Yes, and Fyn restored Cr(VI)-stimulated STAT1 tyrosine phosphorylation and IRF7 expression. Finally, shRNA knockdown of Fyn in BEAS-2B cells prevented Cr(VI)-activated STAT1 transactivation of IRF7. These data support a novel mechanism through which Cr(VI) stimulates Fyn to initiate interferon-like signaling for STAT1-dependent gene transactivation.

Regulation of protein kinase Cδ downregulation by protein kinase Cε and mammalian target of rapamycin complex 2

Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKCδ by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKCδ downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKCδ at the activation loop (Thr505), turn motif (Ser643), hydrophobic motif (Ser662) and Tyr-311 sites to a greater extent in HeLa/CP cells compared to HeLa cells. Prolonged treatment with PDBu led to downregulation of PKCδ in HeLa but not in HeLa/CP cells. The PKC inhibitor Gö 6983 inhibited PDBu-induced downregulation of PKCδ, decreased Thr505 phosphorylation and increased PKCδ tyrosine phosphorylation at Tyr-311 site. However, knockdown of c-Abl, c-Src, Fyn and Lyn had little effect on PKCδ downregulation and Tyr311 phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor Ly294002 and mTOR inhibitor rapamycin restored the ability of PDBu to downregulate PKCδ in HeLa/CP cells. Knockdown of mTOR and rictor but not raptor facilitated PKCδ downregulation. Depletion of PKCε also enhanced PKCδ downregulation by PDBu. These results suggest that downregulation of PKCδ is regulated by PKCε and mammalian target of rapamycin complex 2 (mTORC2).

The Involvement of Fyn Tyrosine Kinase in the Signal Transduction of Ca2+ sensitization of Vascular Smooth Muscle Contraction Mediated by Rho-kinase

Rho-kinase (ROK)-mediated Ca2+ sensitization of vascular smooth muscle (VSM) contraction plays a critical role in abnormal VSM contraction such as vasospasm. Previously we found that sphingosylphosphorylcholine (SPC) induces the ROK-mediated Ca2+ sensitization through the activation of Src family tyrosine kinase (Src-TK) independently of a protein kinase C pathway. Since western blot analysis showed the presence of Fyn and c-Src among Src-TKs in VSM, we aimed to clarify which Src-TK is truly involved in the Ca2+ sensitization mediated by the SPC/ROK pathway. Immunofluorescent study showed that SPC induced the translocation of Fyn, but not c-Src, to plasma membrane in cultured VSM cells, which was blocked by eicosapentaenoic acid, a specific inhibitor of the SPC-induced Ca2+ sensitization. The siRNA-mediated knockdown of Fyn diminished the SPC-induced contraction remarkably in cultured VSM cells. In β-escin-permeabilized VSM strips, constitutively- active Fyn, which was produced by a baculovirus expression system, induced the Ca2+ sensitization, which was blocked by Y27632 (a ROK inhibitor). Dominant-negative Fyn blocked the Ca2+ sensitization induced by SPC, GTPγS (a direct activator of G-protein), and a GPCR agonist + GTP. Overexpression of constitutively-active and dominant-negative Fyn induced contraction and relaxation of cultured VSM cells, respectively. Furthermore, in order to identify the target(s) of Fyn, tyrosine-phosphorylated proteins were immunoprecipitated from the VSM cells which were treated with or without SPC. Subsequently, nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) was used to identify the SPC-induced tyrosine-phosphorylated and tyrosine-dephosphorylated proteins and their phosphorylation sites. These findings clearly indicate that Fyn tyrosine kinase plays an essential role in the ROK-mediated Ca2+ sensitization of VSM contraction. Functional proteomic approach has been useful to reveal possible targets of Fyn, and their functional roles are currently under investigation.

Comparative Phosphoproteomics of Zebrafish Fyn/Yes Morpholino Knockdown Embryos

The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

Kinase requirements in human cells: II. Genetic interaction screens identify kinase requirements following HPV16 E7 expression in cancer cells

Human papillomavirus (HPV) oncoproteins subvert cellular signaling pathways, including kinase pathways, during the carcinogenic process. To identify kinases targeted by the HPV16 E7 oncoprotein, shRNA kinase screens were performed in RKO colorectal carcinoma cell lines that differ only in their expression of HPV16 E7. Our screens identified kinases that were essential for the survival of RKO cells, but not essential for RKO cells expressing HPV16 E7. These kinases include CDK6, ERBB3, FYN, AAK1, and TSSK2. We show that, as predicted, CDK6 knockdown inhibits pRb phosphorylation and induces S-phase depletion, thereby inhibiting cell viability. Knockdown of ERBB3, FYN, AAK1, and TSSK2 induces a similar loss of cell viability through an unknown mechanism. Expression of the HPV16 E7 oncoprotein, known to bind and degrade pRb, relieves the requirement of these kinases. These studies demonstate that expression of a single oncoprotein can dramatically alter kinase sensitivity in human cells. The shRNA screens used here perform analogously to genetic interaction screens commonly used in genetically tractable organisms such as yeast, and thus represent an exciting method for unbiased identification of cellular signaling pathways targeted by cancer mutations.

Role of src-family kinases in hypoxic vasoconstriction of rat pulmonary artery

AimsWe investigated the role of src-family kinases (srcFKs) in hypoxic pulmonary vasoconstriction (HPV) and how this relates to Rho-kinase-mediated Ca2+ sensitization and changes in intracellular Ca2+ concentration ([Ca2+]i).Methods and resultsIntra-pulmonary arteries (IPAs) were obtained from male Wistar rats. HPV was induced in myograph-mounted IPAs. Auto-phosphorylation of srcFKs and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and myosin light-chain (MLC20) in response to hypoxia were determined by western blotting. Translocation of Rho-kinase and effects of siRNA knockdown of src and fyn were examined in cultured pulmonary artery smooth muscle cells (PASMCs). [Ca2+]i was estimated in Fura-PE3-loaded IPA. HPV was inhibited by two blockers of srcFKs, SU6656 and PP2. Hypoxia enhanced phosphorylation of three srcFK proteins at Tyr-416 (60, 59, and 54 kDa, corresponding to src, fyn, and yes, respectively) and enhanced srcFK-dependent tyrosine phosphorylation of multiple target proteins. Hypoxia caused a complex, time-dependent enhancement of MYPT-1 and MLC20 phosphorylation, both in the absence and presence of pre-constriction. The sustained component of this enhancement was blocked by SU6656 and the Rho-kinase inhibitor Y27632. In PASMCs, hypoxia caused translocation of Rho-kinase from the nucleus to the cytoplasm, and this was prevented by anti-src siRNA and to a lesser extent by anti-fyn siRNA. The biphasic increases in [Ca2+]i that accompany HPV were also inhibited by PP2.ConclusionHypoxia activates srcFKs and triggers protein tyrosine phosphorylation in IPA. Hypoxia-mediated Rho-kinase activation, Ca2+ sensitization, and [Ca2+]i responses are depressed by srcFK inhibitors and/or siRNA knockdown, suggesting a central role of srcFKs in HPV.

TLR4 Signaling Is Coupled to SRC Family Kinase Activation, Tyrosine Phosphorylation of Zonula Adherens Proteins, and Opening of the Paracellular Pathway in Human Lung Microvascular Endothelia

Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein-tyrosine kinases (PTKs) and their substrates required for LPS-induced protein tyrosine phosphorylation and opening of the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls). LPS disrupted barrier integrity in a dose- and time-dependent manner, and prior broad spectrum PTK inhibition was protective. LPS increased tyrosine phosphorylation of zonula adherens proteins, VE-cadherin, gamma-catenin, and p120(ctn). Two SRC family PTK (SFK)-selective inhibitors, PP2 and SU6656, blocked LPS-induced increments in tyrosine phosphorylation of VE-cadherin and p120(ctn) and paracellular permeability. In HMVEC-Ls, c-SRC, YES, FYN, and LYN were expressed at both mRNA and protein levels. Selective small interfering RNA-induced knockdown of c-SRC, FYN, or YES diminished LPS-induced SRC Tyr(416) phosphorylation, tyrosine phosphorylation of VE-cadherin and p120(ctn), and barrier disruption, whereas knockdown of LYN did not. For VE-cadherin phosphorylation, knockdown of either c-SRC or FYN provided total protection, whereas YES knockdown was only partially protective. For p120(ctn) phosphorylation, knockdown of FYN, c-SRC, or YES each provided comparable but partial protection. Toll-like receptor 4 (TLR4) was expressed both on the surface and intracellular compartment of HMVEC-Ls. Prior knockdown of TLR4 blocked both LPS-induced SFK activation and barrier disruption. These data indicate that LPS recognition by TLR4 activates the SFKs, c-SRC, FYN, and YES, which, in turn, contribute to tyrosine phosphorylation of zonula adherens proteins to open the endothelial paracellular pathway.

Receptor for activated C kinase1 (RACK1) binds with G beta1 and restricts focal adhesion kinase activation and restoration of endothelial barrier function

We showed that sustained activation of focal adhesion kinase (FAK) is required for reversing the increase in endothelial permeability induced by inflammatory mediator thrombin. However, mechanism that sustain FAK activation is not known and therefore will help in identifying signaling cues responsible for restoring endothelial barrier function. Gβ1 and receptor for activated C kinase (RACK1) belong to large family of WD40 repeat proteins and act as scaffold proteins to orchestrate downstream signaling events. We therefore investigated the contribution of RACK1 and Gβ1 in regulating FAK activation and recovery of endothelial barrier function. We show that RACK1 is associated with Gβ1 in untreated cells. Thrombin induced a time‐dependent reversible dissociation of RACK1 from Gβ1, enabling it to interact with upstream FAK regulator Fyn. Inhibiting endogenous expression of Gβ1 had no effect on thrombin‐induced increase in endothelial permeability but markedly impaired endothelial barrier repair. Knockdown of Fyn in endothelial cells also prevented barrier recovery post‐thrombin challenge, linking Gβ1 to the regulation of Fyn function. In contrast, knock down of RACK1 increased basal phosphorylation of FAK and potentiated endothelial barrier recovery. Thus, these data demonstrate that RACK1 prevents the access of Gβ1 from forming a complex with Fyn, which is required for FAK activation and restoration of endothelial barrier function.Supported by NIH grants: HL71794 and HL84153

BCR-ABL1 mediates up-regulation of Fyn in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) invariably progresses to blast crisis, which represents the most proliferative phase of the disease. The BCR-ABL1 oncogene stimulates growth and survival pathways by phosphorylating numerous substrates, including various Src family members. Here we describe up-regulation, in contrast to activation, of the ubiquitously expressed Src kinase, Fyn, by BCR-ABL1. In a tissue microarray, Fyn expression was significantly increased in CML blast crisis compared with chronic phase. Cells overexpressing BCR-ABL1 in vitro and in vivo display an up-regulation of Fyn protein and mRNA. Knockdown of Fyn with shRNA slows leukemia cell growth, inhibits clonogenicity, and leads to increased sensitivity to imatinib, indicating that Fyn mediates CML cell proliferation. In severe combined immunodeficient (SCID) mice injected with Fyn shRNA-expressing cells, myeloid-derived cell numbers dropped by 50% and death from leukemia was delayed. Taken together, these results encourage the development of therapies targeting Fyn expression.

Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.

Fyn Upregulation Is a Novel ROS-Dependent Mechanism Controlling CML Growth, Progression and Imatinib Resistance.

The BCR/ABL kinase alters the oxidative environment in chronic myelogenous leukemia (CML) cells, but the consequences of the increased reactive oxygen species (ROS) levels on signaling pathways remain unknown. Increased intracellular peroxides in BCR/ABL expressing cells have been linked to DNA damage, which may promote blast crisis in CML. We report that Fyn is a BCR/ABL target that is upregulated in an oxidant- sensitive manner. Cells overexpressing BCR/ABL display a four-fold upregulation of Fyn protein, which is blocked by chemical antioxidants. This increase in Fyn directs proliferative and survival signals since knockdown of Fyn using shRNA slows leukemia cell growth by 50% both in vitro and in vivo, inhibits clonogenic growth by 45% and leads to increased sensitivity to imatinib. Jak2 inhibition prevents Fyn protein upregulation, suggesting that Jak2 is upstream of Fyn, and we indeed find that Jak2 levels are increased in BCR/ABL expressing cells. In a cohort of CML patients Fyn expression was significantly increased in blastic phase CML samples as compared to chronic phase, confirming the clinical relevance of Fyn upregulation. Collectively, these results demonstrate that oxidant-dependent, Jak2-dependent upregulation of Fyn is a novel alteration in CML that is critical for cell growth and imatinib resistance.

Essential role for Csk upstream of Fyn and Yes in zebrafish gastrulation

Morphogenetic cell movements during gastrulation shape the vertebrate embryo bodyplan. Non-canonical Wnt signaling has been established to regulate convergence and extension cell movements that mediate anterior-posterior axis elongation. In recent years, many other factors have been implicated in the process by modulation of non-canonical Wnt signaling or by different, unknown mechanisms. We have found that the Src family kinases, Fyn and Yes, are required for normal convergence and extension cell movements in zebrafish embryonic development and they signal in parallel to non-canonical Wnts, eventually converging on a common downstream factor, RhoA. Here, we report that Csk, a negative regulator of Src family kinases has a role in gastrulation cell movements as well. Csk knock down induced a phenotype that was similar to the defects observed after knock down of Fyn and Yes, in that gastrulation cell movements were impaired, without affecting cell fate. The Csk knock down phenotype was rescued by simultaneous partial knock down of Fyn and Yes. We conclude that Csk acts upstream of Fyn and Yes to control vertebrate gastrulation cell movements.